In most eucaryotic genes, a common set of nucleotides has been found before the start site of RNA transcription. These nucleotides, referred to as either a TATA or Goldberg-Hogness box, are important determinants of transcription in vivo and in vitro. The control region for the late SV40 transcripts have been examined by site specific mutagenesis and by generation of deletion mutations. Mutations that enhance or suppress the tanscriptional activity of a single start site have been identified and differ from the consensus Goldberg-Hogness box. The presence in SV40 of a weaker late promoter as compared with the early promoter may be a mechanism to regulate genes in a temporal manner. By late genes having a lower affinity for RNA polymerase II, the synthesis of early transcripts that are required to initiate viral DNA replication are favored. An additional cluster of nucleotides that is located at base positions 75-96 also enhances the level of transcription and it maintains this enhancing effect when its position relative to the 5' start site is altered.